Week 1

Thu Jun 15

Transformation of standard components

ligation and transformation of promoter and GFP

ligation protocol with the Roche Rapid DNA ligation kit:

6 μL of insert (E0241), 1 μL of plasmid (R0010 and backbone), and 3 μL of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 μL) into a microcentrifuge tube. (Would ordinarily use 2 μL of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)

Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.

digested promoter (R0010) and GFP (E0241) plasmids

R0010 digested with SpeI and PstI in order to leave it attached at upstream end to the plasmid backbone (otherwise, the fragment would only be ~200 bp long, which is a little too short for electrophoresis with much longer fragments

E0241 digested with XbaI and PstI in order to cleave it as a fragment

The following ingredients were each added to four 1.5 ml centrifuge tubes:

Mon Jun 12

goal: insert three BioBrick plasmids (already containing BioBricks) into E. coli in order to amplify them

a positive control (E7104) with the T7 promoter upstream of GFP

the lac operon (R0010)

GFP (E0241)

transformation protocol:

1 μL each of E7104, R0010, E0241, and water into separate microcentrifuge tubes that each contain 30 μL of competent cells

Incubated the tubes on ice for 20 min.

Heat shocked the tubes at 42°C for 30 s.

Added 200 μL SOC to each tube.

Incubated, with shaking, at 37°C for 1 h.

Plated the mixture on carbomycin plates and incubated at 37°C for 24 h.

Literature summaries and brainstorming

Shih's 2004 Nature paper (biblio below)

Shih describes the (rather complex) construction of the DNA octahedron, which consists of two motifs: double-crossover edges ("struts") and paranemic crossover struts. Each edge of the octahedron is combrised of two ds DNAs which are interwoven with these motifs. Edges are connected at vertices by two unpaired thymine residues for flexibility. The structure was formed from a long "heavy chain" (scaffold) and five 40-nt "light chains" (oligos) in two steps: first, by cooling the structure from a denaturation temperatue to form a branched complex, and then further cooling to allow corresponding paranemic crossover areas to associate.

The final formation step is proven through gel electrophoresis. Mg2+ is required for the final folding step, and the product runs faster after Mg2+ has been added, indicating further folding. Other confirmatory steps were complicated: the structures were visualized with cryo-electron microscopy, and a 3d model was formed using microscopy images from 961 particles.

Shih points out that no covalent bonds are created or broken in the formation of the structure, which greatly simplifies the assembly process. He also suggests one possible application/implication of his work, which is that one-dimensional information (the primary DNA sequence of the heavy chain) is effectively encoding 3d positional information (i.e., the final 3d position of the nucleotide).

Yurke's 2000 Nature paper (biblio below)

Yurke's diagram of his DNA tweezers

Yurke describes the DNA "tweezers" he engineered, which consist of the following parts:

a short (~30 nt) ss DNA backbone (A)

two oligos (B and C) which are each complimentary to half of the backbone and bound to it, and each of which have a short (~20 nt) ss overhang

In this state, the DNA tweezers are in an "open" conformation. A ~50-nt "fuel strand" of ss DNA (F) is added to the tweezers to close them, and this occurs because the fuel strand is complimentary to each of the oligo overhangs, which closes the tweezers. The fuel strand also includes a 8-nt ss overhang, and so when a strand complimentary to the fuel strand ("displacement" strand") (F-bar) is added, the fuel strand is stripped, opening the tweezers.

Yurke reports that the rate-limiting step of this "strand displacement" is the initial binding between the fuel strand and the displacement strand, and the time required for DNA branch migration (on this scale) is negligble. The tweezers open and shut on a time scale of a few minutes after a fuel (or displacement) strand is added.

A convenient way to open a DNA nanobox would be to construct a box held shut by a latch with a DNA tweezer motif in its closed conformation, such that a displacement strand would open the box. Yurke opens his tweezers through the exogenous addition of a displacement strand, but I suggest that we look for a way to bind some sort of ss DNA to the surface of a cell or cellular protein, so that when a nanobox approaches, the strand would unhook the latch.

Given the (hypothesized) large amount of cellular protrusions from E. coli, this could be difficult to engineer, and there may be a kinetic difficulty with the constraining of the displacement strand.

Yurke carried out the reaction in at 20°C SPSC buffer, which contains 50mM sodium biphosphate, 1M NaCl, and pH 6.5. We should investigate if strand displacement will work at physiological pH and lower salt concentrations as well.